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Thesis presented by ���������������������� ����� � �
M.Sc. in Pharmaceutical Sciences (Pharmaceutical Chemistry)
Faculty of Pharmacy Cairo University
(2007)
Submitted for the fulfillment of Ph.D. Degree in Pharmaceutical Sciences
(Pharmaceutical Chemistry)
Under the supervision of
Prof. Dr. Fatma Abd El-Fattah Ragab
Professor of Pharmaceutical Chemistry Pharmaceutical Chemistry Department
Faculty of Pharmacy Cairo University
Prof. Dr. Mostafa Mohamed Ghorab
Professor of Applied Organic Chemistry Drug Radiation Research Department
National Centre for Radiation Research and Technology
Atomic Energy Authority
Dr. Helmy Ismail Heiba
Assoc. Professor of Applied Organic Chemistry
Drug Radiation Research Department National Centre for Radiation Research and
Technology Atomic Energy Authority
Dr. Reem Khidr Arafa
Lecturer of Pharmaceutical Chemistry Pharmaceutical Chemistry Department
Faculty of Pharmacy Cairo University
Faculty of Pharmacy
Cairo University (2010)
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Synthesis of some new heterocyclic
compounds bearing a sulfonamide moiety and studying their combined anticancer
effect with �-radiation Presented by: Ebaa Mostafa Mohamed El-Hossary Approved by: Prof. Dr. Fatma Abdel-Fattah Ragab .................................... Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University Dr. Helmy Ismail Heiba .................................... Assoc. Professor of Applied Organic Chemistry National Centre for Radiation Research & Technology Prof. Dr. Hassan Hassan Farag .................................... Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Assuit University Prof. Dr. Kamelia Mahmoud Amin .................................... Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University
AcknowledgementAcknowledgementAcknowledgementAcknowledgement Firstly, I want to thank Allah Allah Allah Allah who gave me the patience and support to achieve my goal. Many thanks to Prof. Dr. Fatma Abd ElProf. Dr. Fatma Abd ElProf. Dr. Fatma Abd ElProf. Dr. Fatma Abd El----Fattah RagabFattah RagabFattah RagabFattah Ragab, Prof. of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, for her encouragement and kind supervision. Words couldn’t help me in expressing my deep appreciation to Prof. Prof. Prof. Prof. Dr.Dr.Dr.Dr. Mostafa MMostafa MMostafa MMostafa Mohamedohamedohamedohamed Ghorab, Ghorab, Ghorab, Ghorab, Professor of Applied Organic Chemistry, Drug Radiation Research Department, National Centre for Radiation Research and Technology, for suggesting the research project and his great support, faithful advice and unlimited efforts in this research. My special thanks to Dr. Helmy Ismail HeibaDr. Helmy Ismail HeibaDr. Helmy Ismail HeibaDr. Helmy Ismail Heiba, Assoc. Prof. of Applied Organic Chemistry, National Centre for Radiation Research and Technology, for his great support, helpful suggestions and advice which helped me to reach this work in its final form. I am also grateful to Dr. Reem Khidr Arafa, Dr. Reem Khidr Arafa, Dr. Reem Khidr Arafa, Dr. Reem Khidr Arafa, Lecturer of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, for her helpful suggestions and advice which helped me to finalize this work. I would like to give my special thanks to my whole famfamfamfamilyilyilyily for the patience and moral support they provided through out my research work. Finally, my deep thanks to my colleagues my colleagues my colleagues my colleagues and to all who gave me help and support for the fulfillment of this work.
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Contents
Abstract……………………………………...…………………... I - VII
1. Introduction…………………………………………...……… 1
1.1. Sulfonamides as anticancer agents………………………… 1
1.2. Quinolines as anticancer agents…….……………………… 16
1.3. Chemoradiotherapy………………………………………… 19
1.4. Synthesis of quinolines………………………...….….......... 25
1.5. Synthesis of pyrimido[4,5-b]quinolines…………………… 34
2. Aim of the present investigation……………………...…....... 38
3. Theoretical Discussion……………………………...……....... 43
4. Experimental…………………………………………………. 75
5. Biological Activity………………………………...………….. 117
5.1. In vitro anticancer screening……………………………….. 117
5.2. Radiosensitizing evaluation……………...…….…………... 138
6. Molecular Docking………………………………….....……... 142
7. References…………………………………...……………....... 150
Arabic summary………………………………..………………. �
List of Figures
Figure (1): Schematic representation of the catalytic mechanism for the �-CA catalysed CO2 hydration… … … … … … … … … .. 3
Figure (2): Zinc binding groups… … … … … … … … … … … … … … … 4 Figure (3): Structural elements of CA inhibitors in the CA enzymatic
active site… … … … … … … … … … … … … … … … … … … . 4 Figure (4): The cell cycle… … … … … … … … … … … … … … … … … .. 11 Figure (5): Dose-response curves for tumor control and normal tissue
damage… … … … … … … … … … … … … … … … … … … … . 19 Figure (6): Representative examples of the synthesized compounds
showing great compliance to the general pharmacophore of CA inhibitors… … … … … … … … … … … … … … … … ... 40
Figure (7): Survival curve of Doxorubicin… … … … … … … … … … … 119 Figure (8): Survival curve of compound 6a… … … … … … … … … … .. 119 Figure (9): Survival curve of compound 6b… … … … … … … … … … .. 119 Figure (10): Survival curve of compound 7a… … … … … … … … … … 120 Figure (11): Survival curve of compound 7b… … … … … … … … … … 120 Figure (12): Survival curve of compound 8a… … … … … … … … … … 120 Figure (13): Survival curve of compound 8b… … … … … … … … … … 121 Figure (14): Survival curve of compound 9a… … … … … … … … … … 121 Figure (15): Survival curve of compound 9b… … … … … … … … … … 121 Figure (16): Survival curve of compound 10a… … … … … … … … … .. 122 Figure (17): Survival curve of compound 10b… … … … … … … … … .. 122 Figure (18): Survival curve of compound 11a… … … … … … … … … .. 122 Figure (19): Survival curve of compound 11b… … … … … … … … … .. 123 Figure (20): Survival curve of compound 12a… … … … … … … … … .. 123 Figure (21): Survival curve of compound 12b… … … … … … … … … .. 123 Figure (22): Survival curve of compound 13a… … … … … … … … … .. 124 Figure (23): Survival curve of compound 13b… … … … … … … … … .. 124 Figure (24): Survival curve of compound 14a… … … … … … … … … .. 124 Figure (25): Survival curve of compound 14b… … … … … … … … … .. 125 Figure (26): Survival curve of compound 15a… … … … … … … … … .. 125 Figure (27): Survival curve of compound 15b… … … … … … … … … .. 125 Figure (28): Survival curve of compound 16a… … … … … … … … … .. 126 Figure (29): Survival curve of compound 16b… … … … … … … … … .. 126 Figure (30): Survival curve of compound 17a… … … … … … … … … .. 126
Figure (31): Survival curve of compound 17b… … … … … … … … … .. 127 Figure (32): Survival curve of compound 18a… … … … … … … … … .. 127 Figure (33): Survival curve of compound 18b… … … … … … … … … .. 127 Figure (34): Survival curve of compound 19a… … … … … … … … … .. 128 Figure (35): Survival curve of compound 19b… … … … … … … … … .. 128 Figure (36): Survival curve of compound 20a… … … … … … … … … .. 128 Figure (37): Survival curve of compound 20b… … … … … … … … … .. 129 Figure (38): Survival curve of compound 21a… … … … … … … … … .. 129 Figure (39): Survival curve of compound 21b… … … … … … … … … .. 129 Figure (40): Survival curve of compound 22a… … … … … … … … … .. 130 Figure (41): Survival curve of compound 22b… … … … … … … … … .. 130 Figure (42): Survival curve of compound 23a… … … … … … … … … .. 130 Figure (43): Survival curve of compound 23b… … … … … … … … … .. 131 Figure (44): Survival curve of compound 24a… … … … … … … … … .. 131 Figure (45): Survival curve of compound 24b… … … … … … … … … .. 131 Figure (46): Survival curve of compound 25a… … … … … … … … … .. 132 Figure (47): Survival curve of compound 25b… … … … … … … … … .. 132 Figure (48): Survival curve for MCF7 cell line for compound 8b
alone or in combination with �-irradiation (8 Gy)… … … 140 Figure (49): Survival curve for MCF7 cell line for compound 11a
alone or in combination with �-irradiation (8 Gy)… … … 140 Figure (50): Survival curve for MCF7 cell line for compound 12a
alone or in combination with �-irradiation (8 Gy)… … … 141 Figure (51): Survival curve for MCF7 cell line for compound 18b
alone or in combination with �-irradiation (8 Gy)… … … 141 Figure (52): Superimposition of hCA II-inhibitor adducts… … … … ... 142 Figure (53): CA inhibition mechanism by sulfonamides… … … … … .. 143 Figure (54): Interaction map of N-(2,3,4,5,6-pentafluorobenzyl)-4-
sulfamoyl-benzamide with hCA II… … … … … … … … … 144 Figure (55): Interaction map of E7070 with the active site of hCA II.. 145 Figure (56): Interaction map of compound 8b with the active site of
hCA II… … … … … … … … … … … … … … … … … … … ... 146 Figure (57): Interaction map of compound 11a with the active site of
hCA II… … … … … … … … … … … … … … … … … … … .. 147 Figure (58): Interaction map of compound 12a with the active site of
hCA II… … … … … … … … … … … … … … … … … … … ... 147 Figure (59): Interaction map of compound 18b with the active site of
hCA II… … … … … … … … … … … … … … … … … … … ... 148 Figure (60): Superimposition of E7070 and compound 11a in the
active site of hCA II… … … … … … … … … … … … … … . 149
List of Tables
Table (1): Physical data and microanalysis of compounds 6a & 6b… … … . 77 Table (2): Physical data and microanalysis of compounds 7a & 7b… … … . 79 Table (3): Physical data and microanalysis of compounds 8a & 8b… … … . 81 Table (4): Physical data and microanalysis of compounds 9a & 9b… … … . 83 Table (5): Physical data and microanalysis of compounds 10a & 10b… … . 85 Table (6): Physical data and microanalysis of compounds 11a & 11b… … . 87 Table (7): Physical data and microanalysis of compounds 12a & 12b… … . 89 Table (8): Physical data and microanalysis of compounds 13a & 13b… … . 91 Table (9): Physical data and microanalysis of compounds 14a & 14b… … . 93 Table (10): Physical data and microanalysis of compounds 15a & 15b… ... 95 Table (11): Physical data and microanalysis of compounds 16a & 16b… ... 97 Table (12): Physical data and microanalysis of compounds 17a & 17b… ... 99 Table (13): Physical data and microanalysis of compounds 18a & 18b… ... 101 Table (14): Physical data and microanalysis of compounds 19a & 19b… ... 103 Table (15): Physical data and microanalysis of compounds 20a & 20b… ... 105 Table (16): Physical data and microanalysis of compounds 21a & 21b… ... 107 Table (17): Physical data and microanalysis of compounds 22a & 22b… ... 109 Table (18): Physical data and microanalysis of compounds 23a & 23b… ... 111 Table (19): Physical data and microanalysis of compounds 24a & 24b… ... 113 Table (20): Physical data and microanalysis of compounds 25a & 25b… ... 115 Table (21): In vitro anticancer screening of the synthesized compounds
against human breast cancer cell line (MCF7)… … .… … … … ... 133 Table (22): In vitro anticancer screening of compounds 8b, 11a, 12a and
18b against human breast cancer cell line (MCF7) in combination with �-radiation… … … ...… … … … … … … … … ... 139
Abbreviations
• ANOVA: Analysis of variance.
• CA: Carbonic anhydrase.
• CAI: Carbonic anhydrase inhibitor.
• CARP: Carbonic anhydrase related protein.
• CDK: Cyclin dependent kinase.
• CNS: Central nervous system.
• Conc.: concentrated.
• CRT: Chemoradiotherapy.
• CT: Chemotherapy.
• Cys: Cysteine.
• dil.: diluted.
• DMF: dimethyl formamide.
• DMSO: Dimethyl sulfoxide.
• DNA: Deoxyribonucleic acid.
• EDTA: Ethylene diamine tetra-acetic acid.
• EGFR: Epidermal growth factor receptor.
• EI/MS: Electron impact mass spectrometry.
• ELISA: Enzyme-linked immunosorbent assay.
• 5-FU: 5-fluorouracil.
• Gln: Glutamine.
• Glu: Glutamic acid.
• h: hour.
• hCA: human carbonic anhydrase.
• His: Histidine.
• 1H-NMR: Proton nuclear magnetic resonance.
• IR: Infrared.
• Leu: Leucine.
• MDR: Multi-drug resistant.
• min: minute.
• MMFF94x: Merck Molecular Force Field 94x.
• MMP: Matrix metalloproteinase.
• MMPIs: Matrix metalloproteinase inhibitors.
• MOE: Molecular operating environment.
• MWI: Microwave irradiation.
• NSCLC: Non-small cell lung cancer.
• OTT: Overall treatment time.
• Phe: Phenylalanine.
• PI3: Phosphoionositide 3.
• pRb: Retinoblastoma protein.
• Pro: Proline.
• PTSA: p-Toluene sulfonic acid.
• RNA: Ribonucleic acid.
• RT: Radiotherapy.
• SAR: Structure activity relationship.
• SE: Standard error.
• SRB: Sulfo-rhodamine B.
• TCA: Trichloroacetic acid.
• TEA: Triethylamine.
• TFAE: Trifluoro-acetaldehyde ethyl hemiacetal.
• Thr: Threonine.
• TLC: Thin layer chromatography.
• TMS: Tetramethylsilane.
• UK: United Kingdom.
• USA: United States of America.
• UV: Ultraviolet.
• Val: Valine.
• ZBG: Zinc binding group.
Abstract
I
In search for new cytotoxic agents with improved anticancer profile, some
new halogen-containing quinoline and pyrimido[4,5-b]quinoline derivatives
bearing a free sulfonamide moiety were synthesized. All the newly synthesized
target compounds were subjected to in vitro anticancer screening against human
breast cancer cell line (MCF7). The most potent compounds, as concluded from
the in vitro anticancer screening, were selected to be evaluated again for their in
vitro anticancer activity in combination with �-radiation. Also, the newly
synthesized compounds were docked in the active site of the carbonic anhydrase
enzyme.
The thesis includes the following parts:
1. Introduction
This part includes a brief literature review on anticancer activity of
sulfonamides and quinoline derivatives regarding their mechanisms of action, and
the rationale for combining chemotherapy and radiotherapy. In addition, the
different methods for the synthesis of quinoline derivatives and pyrimido[4,5-
b]quinoline derivatives are discussed.
2. Aim of the present investigation
The rationale upon which synthesis of the new compounds, evaluation of their
anticancer activity alone or in combination with �-irradiation, and suggestion of
their mechanism of action through a docking study, is presented in this part.
3. Theoretical discussion
This part deals with the discussion of the experimental methods adopted for
the synthesis of the synthesized compounds, as well as different analytical
methods adopted for the identification and the verification of the structures of the
Abstract
II
synthesized compounds. Schemes (1-5) illustrate the synthetic pathways adopted
in the preparation of the designed compounds.
4. Experimental
This part describes the practical procedures used for the synthesis of forty
new final compounds and one known intermediate, with their elemental analyses
and spectral data (IR, 1H-NMR and mass spectroscopy).
Known intermediate:
• 4-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)benzenesulfonamide (3)
New final compounds:
• 4-[2-Amino-3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (6a)
• 4-[2-Amino-4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (6b)
• 4-[5-(4-Fluorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (7a)
• 4-[5-(4-Chlorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (7b)
• N-[3-Cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (8a)
Abstract
III
• N-[4-(4-Chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (8b)
• N-Acetyl-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9a)
• N-Acetyl-N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-
sulfamoyl-phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9b)
• 4-[5-(4-Fluorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (10a)
• 4-[5-(4-Chlorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (10b)
• 4-[4-Amino-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (11a)
• 4-[4-Amino-5-(4-chlorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (11b)
• Ethyl N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12a)
• Ethyl N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12b)
Abstract
IV
• 2-Chloro-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (13a)
• 2-Chloro-N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-
sulfamoyl-phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (13b)
• 4-[2-(Chloromethyl)-5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl]benzenesulfonamide (14a)
• 4-[2-(Chloromethyl)-5-(4-chlorophenyl)-8,8-dimethyl-4,6-dioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl]benzenesulfonamide (14b)
• 4-[2-(Cyanomethyl)-5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl]benzenesulfonamide (15a)
• 4-[5-(4-Chlorophenyl)-2-(cyanomethyl)-8,8-dimethyl-4,6-dioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl]benzenesulfonamide (15b)
• 4-[3-Cyano-2-(3-ethylthioureido)-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-
5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (16a)
• 4-[4-(4-Chlorophenyl)-3-cyano-2-(3-ethylthioureido)-7,7-dimethyl-5-oxo-
5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (16b)
Abstract
V
• 4-[3-Cyano-2-(2,5-dioxopyrrolidin-1-yl)-4-(4-fluorophenyl)-7,7-dimethyl-
5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (17a)
• 4-[4-(4-Chlorophenyl)-3-cyano-2-(2,5-dioxopyrrolidin-1-yl)-7,7-dimethyl-
5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (17b)
• 2-Amino-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-phenyl)-
1,4,5,6,7,8-hexahydroquinoline-3-carboxamide (18a)
• 2-Amino-4-(4-chloro-phenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoylphenyl)-
1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide (18b)
• 4-[2-Amino-3-(4,5-dihydro-1H-imidazol-2-yl)-4-(4-fluorophenyl)-7,7-
dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide
(19a)
• 4-[2-Amino-4-(4-chlorophenyl)-3-(4,5-dihydro-1H-imidazol-2-yl)-7,7-
dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide
(19b)
• N-[3-Cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]-3-oxobutanamide (20a)
• N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]-3-oxobutanamide
(20b)
Abstract
VI
• 4-[5-(4-Fluorophenyl)-8,8-dimethyl-6-oxo-4-thioxo-3,4,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (21a)
• 4-[5-(4-Chlorophenyl)-8,8-dimethyl-6-oxo-4-thioxo-3,4,6,7,8,9-
hexahydro-pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (21b)
• 4-[4-Chloro-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (22a)
• 4-[4-Chloro-5-(4-chlorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (22b)
• Ethyl 2-[5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-10-(4-sulfamoyl-
phenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-3(4H,5H,10H)-
yl]acetate (23a)
• Ethyl 2-[5-(4-chlorophenyl)-8,8-dimethyl-4,6-dioxo-10-(4-
sulfamoylphenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-
3(4H,5H,10H)-yl]acetate (23b)
• 4-(5-[4-Fluorophenyl)-4-hydrazinyl-8,8-dimethyl-6-oxo-6,7,8,9-
tetrahydro-pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (24a)
• 4-[5-(4-Chlorophenyl)-4-hydrazinyl-8,8-dimethyl-6-oxo-6,7,8,9-
tetrahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (24b)
• 4-[5-(4-Fluorophenyl)-4-isothiocyanato-8,8-dimethyl-6-oxo-6,7,8,9-
tetrahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (25a)
Abstract
VII
• 4-[5-(4-Chlorophenyl)-4-isothiocyanato-8,8-dimethyl-6-oxo-6,7,8,9-
tetrahydro-pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (25b)
5. Biological activity
Forty new synthesized compounds were evaluated for their in vitro
anticancer activity against human breast cancer cell line (MCF7), alone or in
combination with �-irradiation. The results are presented and discussed.
6. Molecular Docking
This part includes the docking of the synthesized compounds in the active
site of carbonic anhydrase enzyme to give an idea if these compounds could act
as carbonic anhydrase inhibitors or not as this may have a role in their anticancer
activity.
7. References
This part includes 137 references.
INTRODUCTION
Introduction
1
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1.1. Sulfonamides as anticancer agents
Sulfonamides constitute an important class of drugs, with several types of
pharmacological activities including antibacterial,1 anti-carbonic anhydrase,2
diuretic,3 hypoglycemic4 and antithyroid activity.5 Also, some structurally novel
sulfonamide derivatives have recently been reported to show substantial
antitumor activity in vitro and/or in vivo.
E7010 I, ER-34410 II and E7070 (Indisulam) III are examples for
antitumor sulfonamides in advanced clinical trials.6
There are a variety of mechanisms describing the antitumor action of
sulfonamides, such as carbonic anhydrase (CA) inhibition, cell cycle arrest in the
G1 phase, disruption of microtubules and angiogenesis (matrix metalloproteinase,
MMP) inhibition. The most prominent mechanism was the inhibition of carbonic
anhydrase isozymes (CAs).6
Introduction
2
1.1.1. Sulfonamides as carbonic anhydrase inhibitors
1.1.1.1. Carbonic anhydrases
The carbonic anhydrases are metalloenzymes containing zinc ion in their
active site. Carbonic anyhdrases are present in prokaryotes and eukaryotes, and
are encoded by four distinct gene families: the �-CAs, �-CAs, �-CAs and �-CAs.
In mammals the �-CA is present and 16 different �-CA isozymes or CA-related
proteins (CARP) were described with different subcellular localization and tissue
distribution.7, 8 Basically there are several cytosolic forms (CA I-III, CA VII and
CA XIII), five membrane-bound isozymes (CA IV, CA IX, CA XII, CA XIV and
CA XV), two mitochondrial isoforms (CA VA & VB) and CA VI is secreted in
the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII,
CARP X and CARP XI).9
All these CAs are able to catalyze the hydration of CO2 to bicarbonate at
physiological pH (Figure 1).2, 9, 10 This chemical interconversion is crucial since
bicarbonate is the substrate for several carboxylation steps in a number of
fundamental metabolic pathways such as gluconeogenesis, biosynthesis of
several amino acids, lipogenesis, ureagenesis and pyrimidine synthesis.11 Apart
from these biosynthetic reactions, some of the CAs are involved in many
physiological processes related to respiration and transport of CO2/bicarbonate
between metabolizing tissues and the lungs, pH homeostasis and electrolyte
secretion in a variety of tissues/organs.2, 10
Introduction
3
Figure (1): Schematic representation of the catalytic mechanism for the �-CA
catalysed CO2 hydration. The hydrophobic pocket for the binding of substrate(s) is shown schematically at step (B)
1.1.1.2. Carbonic anhydrases inhibition
CA inhibitors are mainly used as antiglaucoma agents,3, 10, 12 anti-thyroid
drugs,12 hypoglycemic agents4 and, ultimately, some novel types of anticancer
agents.13 Sulfonamides are known to possess high affinity for CAs as such
compounds possess a zinc binding group (ZBG) by which they interact with the
metal ion in the active site of the enzyme and the residues Thr 199 and Glu 106
in its neighborhood.14 In recent years, an entire range of new ZBGs were reported
as shown in figure 2. These new ZBGs include, in addition to the classical
sulfonamides, sulfamates, sulfamides, substituted sulfonamide, Schiff’s base,
urea and hydroxyurea derivatives, as well as hydroxamates.8, 14
Introduction
4
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Figure (2): Zinc binding groups: sulfonamides, sulfamates, sulfamides, substituted sulfonamides, Schiff’s base, urea, hydroxyurea and hydroxamates.
A general pharmacophore (Figure 3)15 for the sulfonamide compounds
acting as carbonic anhydrase inhibitors has been reported by Thiry et al. This
pharmacophore originated from the analysis of the CAs active site and from the
structure of inhibitors described in literature.16
Figure (3): Structural elements of CA inhibitors in the CA enzymatic active site.
Introduction
5
1.1.1.3. Role of carbonic anhydrases in cancer
The importance of this family of enzymes for the uptake of bicarbonate by
many organisms, and the presence of a large number of isoforms (which can be
distinguished from each other in activity and are located in different areas inside
the cell) make the CAs undoubtedly involved in cell growth. Furthermore, at least
three CA isozymes (CA IX, CA XII and CA XIV) have close connections with
tumors.11, 17 The role of CAs in cancer can be explained in light of the metabolic
processes required by growing cancer cells that develop with a higher rate of
replication than normal cells. Such a circumstance requires a high flux of
bicarbonate into the cell in order to provide substrate for the synthesis of either
nutritionally essential components (nucleotides) or cell structural components
(membrane lipids).11
1.1.1.4. The action of sulfonamides as anticancer agents through CA inhibition
Sulfonamides CA inhibitors reduce the provision of bicarbonate for the
synthesis of nucleotides (mediated by carbamoyl phosphate synthetase II) and
other cell components such as membrane lipids (mediated by pyruvate
carboxylase). Such mechanism would likely involve CA II and CA V.18
An alternative, or additional mechanism, may involve the acidification of
intracellular milieu as a consequence of CA inhibition by these potent CA
inhibitors.19 It is also possible that the sulfonamides interfere with the activity of
the CA isozymes known to be present predominantly in tumor cells, CA IX, XII
and XIV.2, 10, 17 A combination of these mechanisms proposed above is also
possible.
Introduction
6
1.1.1.5. Examples of sulfonamides acting as anticancer agents by CA inhibition
Several potent, clinically used sulfonamide CA inhibitors such as
acetazolamide IV, methazolamide V, or ethoxzolamide VI have shown to inhibit
the growth of human lymphoma cells.18
A program of screening several hundred sulfonamide CA inhibitors (both
aromatic as well as heterocyclic derivatives) for their tumor cell growth
inhibitory effects against the panel of 60 cancer cell lines of the National Cancer
Institute of the USA, has identified derivatives VII-XI as interesting leads. These
derivatives showed potent activities, against a wide variety of cancer cell lines
including leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS,
renal, prostate and breast cancer cell lines.20, 21
Introduction
7
1.1.1.6. Examples of Sulfonamides acting as selective CA inhibitiors
Several miscellaneous sulfonamides have been synthesized and screened by
Vullo et al.22 in an attempt to obtain selective CA inhibitors especially towards
the tumor-associated transmembrane CA IX and CA XII. From these compounds,
compounds XII-XIV were found to behave as very potent CA IX inhibitors,
although they are weak or medium-weak inhibitors of CA I, II and IV.
Casey et al.23 prepared a series of positively charged, membrane-
impermeant sulfonamide CA inhibitors XV-XVIII and showed high affinity for
the cytosolic isozymes CA I and CA II, as well as for the membrane-bound ones
CA IV and CA IX. As for the aminobenzolamide derivatives XV, it was seen that
the best CA IX inhibitors should contain either only small, compact aliphatic
Introduction
8
moieties substituting the pyridinium ring or a 4-phenyl moiety. On the other
hand, the SAR of the derivatives of compounds XVI-XVIII has shown that for a
given substitution pattern of the pyridinium ring, the activity decreases by
decreasing the spacer (n) between the pyridinium and the benzenesulfonamide
moieties. Compounds XIX and XX are the most potent CA IX inhibitors in this
group of compounds.23, 24
Ex vivo studies showed that this new class of inhibitors is able to
discriminate between the membrane-bound versus the cytosolic isozymes. These
compounds were proved to be unable to penetrate through the membranes,
obviously due to their cationic nature. These compounds constitute the basis of
selectively inhibiting only the target, tumor-associated CA IX in vivo, whereas
the cytosolic isozymes would remain unaffected.23
Polyfluorinated CAIs have shown very good inhibitory properties against
different CA isozymes,25 but such compounds have not been tested for their
interaction with the transmembrane, tumor associated isozyme CA IX. Also there
Introduction
9
was a problem related to the pentafluorophenyl-containing CAIs previously
reported,25 which is the high reactivity of the fluorine atom in para to the
sulfonamide/carboxamido moiety, which was shown to covalently bind to thiol
reagents, such as Cys 239 of �-tubulin, glutathione, cysteine itself, etc., leading
to modification of the thiol reagent/protein.26-28
In order to prepare fluorine-containing CAIs devoid of enhanced reactivity,
miscellaneous derivatives lacking the para-fluorine reactive group, with two
types of such derivatives being prepared: the 2,3,5,6-tetrafluorophenyl-
carboxamides, and the 2,3,5,6-tetrafluoro-phenyl-sulfonamides. All these
compounds showed potent CA II and CA IX inhibition than CA I. Among these,
the first subnanomolar and rather selective CA IX inhibitor has been discovered,
as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide XXI showing an
inhibition constant of 0.8nM against hCA IX, and a selectivity ratio of 26.25
against CA IX over CA II.29
The inhibition of a newly cloned hCA XII – the second tumor-associated
CA isozyme described, after CA IX – has been investigated by Vullo et al.29 with
a miscellaneous series of sulfonamides. CA XII is present in a rather wide range
of tumors and it appears probable that inhibition of this isozyme with potent and
possibly specific inhibitors may have clinical relevance for the development of
novel antitumor therapies. The most selective hCA XII over hCA II inhibitors
were compounds XII, XXII and XXIII.
Introduction
10
A series of indanesulfonamide derivatives XXIV and XXV were
synthesized by Thiry et al.15 and tested for their inhibitory activity against CA IX,
and against CA I and II and two other physiologically relevant CA isozymes, to
measure the selectivity of the compounds. Nearly all the derivatives show a high
inhibitory potency against CA IX and CA II and a weak inhibition against CA I.
Compounds XXVI-XXIX are selective against CA IX with reference to CA I,
while they preferentially inhibit CA IX with reference to CA II.
Introduction
11
1.1.2. Sulfonamides targeting G1 phase of cell cycle
G1 phase of the cell cycle is an important period where various complex
signals interact to decide a cell’s fate: proliferation, quiescence, differentiation, or
apoptosis (Figure 4).30
Figure (4): The cell cycle
It is now well-recognized that malfunctioning of cell cycle control in G1
phase is among the most critical molecular bases for tumorigenesis and tumor
progression. Thus, there is a growing possibility that a small molecule targeting
the control machinery in G1 phase can be a new type of drug efficacious against
refractory clinical cancers.31
E7070 III was found to block the entry of human NSCLC A549 cells into
the S phase, leading to the accumulation of cells in the late G1 phase. In addition,
treatment of A549 cells with E7070 resulted in the inhibition of pRb
phosphorylation, a crucial step in the G1/S transition. Down regulation of CDK2
and cyclin A expressions as well as suppression of CDK2 catalytic activity with
Introduction
12
the induction of p53 and p21, may account for the inhibition of pRb
phosphorylation by E7070.32
In addition, E7070 was shown to inhibit the phosphorylation of CDK2 itself,
leading to the inhibiton of CDK2 catalytic activity. These results suggest that
E7070 may target the late G1 phase of the cell cycle and restore the pRb-
dependent growth-inhibitory pathway disrupted in human NSCLC cells. This is
accomplished by inhibiting CDK2 catalytic activity.32
1.1.3. Sulfonamides causing disruption of microtubules
The effects of E7010 on microtubule structure in colon 38 cells were
examined, and E7010 was shown to cause the disappearance of cytoplasmic
microtubules and mitotic spindles. The experiments clearly demonstrated that the
growth-inhibitory activity of E7010 is caused by the inhibition of microtubule
assembly.33
A novel series of 7-aroylaminoindoline-1-benzenesulfonamides have been
identified by Chang et al.34 as a novel class of highly potent antitubulin agents.
The lead compounds XXX and XXXI exhibit antiproliferative activity, with IC50 values ranging from 8.6 to 11.1 nM in a variety of human cancer cell lines from
different organs, including the MDR-positive cell line. The SAR information of
the 7-aminoindoline-substitution pattern revealed that the 7-amide bond
formation in the indoline-1-sulfonamides contributed to a significant extent for
maximal activity rather than the carbamate, carbonate, urea, alkyl, and
sulfonamide linkers.
Introduction
13
Hu et al.35 have synthesized two series of carbazole sulfonamide
compounds. Compounds such as XXXII and XXXIII exhibit strong activities
against human leukemia cells. Preliminary mode of action studies demonstrated
that the lead compound XXXII arrests tumor cell cycle at M-phase and induces
apoptotic cell death by increasing expression of p53 and promoting bcl-2
phosphorylation. Lead compounds such as XXXII and XXXIII merit further
studies as novel promising antimitotic agents against solid tumors.
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1.1.4. Sulfonamides as matrix metalloproteinase (MMP) inhibitors
The matrix metalloproteinases (MMPs), a family of zinc-containing
endopeptidases, were shown to play a central role in several physiological and
physiopathological processes and in our main interest regards the involvement of
these enzymes in angiogenesis and tumor invasion.36, 37
Introduction
14
At least 20 members of this enzyme family, sharing significant sequence
homology, have been reported. They consist of a Zn(II) ion coordinated by three
histidines, with the fourth ligand being a water molecule/hydroxide ion which is
the nucleophile intervening in the catalytic cycle of the enzyme.38
Inhibition of MMPs is correlated with the coordination of the inhibitor
molecule (in neutral or ionized state) to the catalytic metal ion, with or without
replacement of the metal-bound water molecule.10, 37, 39 Thus, MMP inhibitors
(MMPIs) must contain a zinc-binding function attached to a scaffold that will
interact with other binding regions of the enzymes.38
Sulfonylated amino acid hydroxamates were only recently discovered to act
as efficient MMPIs.40, 41 The first compounds from this class to be developed for
clinical trials are (CGS 27023A) XXXIV and (CGS 25966) XXXV.40 Further
developments in this field have led to some strong and relatively selective
inhibitors of this type such as XXXVI.41 Then a large number of arylsulfonyl
hydroxamates derived from glycine, L-alanine, L-valine and L-leucine possessing
N-benzyl- or N-benzyl-substituted moieties, of types XXXVII, were reported.42
Also, some hydroxamates structurally related to the MMPIs were shown to act as
CAIs XXXVIII.38
Introduction
15
Introduction
16
1.2. Quinolines as anticancer agents
Quinolines and fused quinoline derivatives are known to possess several
biological activities43-49 including anticancer activity.50-57 There are a variety of
mechanisms for the antitumor action of quinolines and fused quinoline
derivatives such as DNA intercalation,58, 59 topoisomerase inhibition,60, 61 cell
cycle arrest,62 and several other mechanisms.
Recently, some quinoline derivatives such as compound XXXIX have been
found to act as PI3 kinase inhibitors. The PI3 kinases are a family of enzymes
displaying a protein kinase activity which mediate signaling pathways having a
central role in a number of cell processes including proliferation and survival.
Deregulation of these pathways is a causative factor in a wide spectrum of human
cancers and other diseases.63
The development of cancer can depend on the accumulation of specific
genetic alterations that allow aberrant cell proliferation, including growth of
tumor cells. Protection from such aberrant growth is provided by several
mechanisms that work by inducing apoptotic cell death in cells undergoing
oncogenic changes. Therefore, for a tumor cell to survive, it must acquire genetic
alterations that perturb the link between abnormal growth and cell death. The p53
tumor suppressor protein can induce apoptotic cell death and plays a pivotal role
Introduction
17
in tumor suppression. The pyrimidoquinoline derivatives such as compound XL
have shown significant antitumor activity by modulating or stabilizing p53
activity.64
Also some reduced quinoline derivatives have shown anticancer activity as
that reported by Liou et al.65 who synthesized tetrahydroquinoline derivatives
such as compound XLI which were evaluated for their antiproliferative activities
against oral epidermoid carcinoma KB cells, non-small lung carcinoma H460
cells, and stomach carcinoma MKN45 cells, as well as one type of MDR-positive
cell line, KB-vin 10 cells. Compound XLI showed significant antiproliferative
activities against the previously mentioned four cell lines, and acting through the
inhibition of tubulin polymerization.
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Introduction
18
Some other tetrahydroisoquinoline derivatives were also found to act as
antiestrogens and antiandrogens such as compound XLII used for treatment of
breast and prostate cancer. It is commonly acknowledged that estrogen and the
estrogen receptors play essential roles in the development of breast tumors,
although the precise mechanisms involved have not been determined. Estrogen
receptors also play a role in prostate cancer, thus agents that modulate estrogen
receptors may also be useful in treatment of prostate cancer. Also in the early
stage of prostate cancer, its growth highly relies on the androgen and the use of
antiandrogen deprivation therapy can strongly slow down the growth rate of
prostate cancer.66
Introduction
19
1.3. Chemoradiotherapy
Chemoradiotherapy (CRT) represents definite progress in clinical oncology.
Recently, the concurrent use of chemotherapy (CT) and radiation therapy (RT)
has become a standard treatment for many types of cancer.
1.3.1. Therapeutic ratio
In general, tumor response and normal tissue damage are positively
correlated with the dose of radiation, and this relationship is commonly described
by a sigmoid curve (Figure 5). The therapeutic ratio is defined as the ratio of the
dose that produces a given probability (50% is most commonly used in
experimental studies) of normal tissue damage and the dose that produces the
same probability of tumor control. When CT is combined with RT, the tumor
control curve shifts to the left, along with the response curve for normal tissue
damage. The goal of combining CT and RT is to obtain a positive therapeutic
ratio, and thus to enhance the antitumor effect while minimizing toxicity to
critical normal tissues.67
Figure (5): Dose-response curves for tumor control and normal tissue damage.
When chemotherapy (CT) is combined with radiotherapy (RT), the tumor control curve shifts to the left (long arrow), and the response curve for normal tissue
damage also shifts in the same direction, as indicated by the short arrow.
Introduction
20
1.3.2. Rationale for combining chemotherapy and radiotherapy
The rationale for combining CT and RT is mainly based on two ideas,68-70
one being spatial cooperation, and the other the enhancement of radiation effects.
Spatial cooperation is effective if CT is sufficiently active to eradicate subclinical
metastases and if the primary local tumor is effectively treated by RT. In this
regard, no interaction between RT and CT is required, but differing toxicities are
needed so that both modalities can be used at effective dosages.
A major limitation is the relatively poor efficacy of anticancer drugs against
common solid tumors in adults. It is often difficult to eradicate even small
subclinical metastases by CT. Also, local failure rates of a primary tumor
following RT are high for many tumor sites.
To decrease the local failure rate, the enhancement of RT effects is
necessary. In the presence of chemotherapeutic drugs, an increased response such
as enhancement occurs within the irradiated volume. However, virtually all
chemotherapeutic agents enhance radiation damage to normal tissues as well.
Consequently, a therapeutic benefit is only achieved if enhancement of the tumor
response is greater than that for normal tissues.
Among the many chemotherapeutic agents used, cisplatin is one of the best
agents for yielding a therapeutic benefit. An enhancing effect by the additional
use of daily cisplatin before each RT fraction was observed in an in vivo animal
study.71
Introduction
21
1.3.3. Mechanisms responsible for CT-RT interactions
Recent clinical trials, including metaanalyses, have shown that CT given
concurrently with RT results in improved local control and survival,72-76 implying
interactions between CT and RT. Five major mechanisms responsible for CT-RT
interactions are discussed in the following paragraphs68-70;
1.3.3.1. Initial radiation damage
The first mechanism responsible for CT-RT interaction is the direct
enhancement of the initial radiation damage, resulting from the incorporation of
the chemotherapeutic drugs into DNA. The primary target for radiation injury is
DNA, where halogenated pyrimidines such as 5-fluorouracil (5-FU) are
incorporated, making the DNA more susceptible to RT. Cisplatin interacts with
nucleophilic sites on DNA or RNA to form intra- and interstrand cross-links.
When cisplatin-DNA cross-links are formed during RT, radioenhancement by
cisplatin may occur. This has been observed in both hypoxic and oxygenated
cells.70
1.3.3.2. Inhibition of radiation damage repair
Secondly, the inhibition of cellular repair increases radiation damage. Cells
have the ability to repair sublethal and potentially lethal radiation damage.68
Halogenated pyrimidines, nucleoside analogs, and cisplatin interfere with cellular
repair mechanisms. This inhibition of cellular repair can be effective when drugs
are administered following fractionated RT. In general, nucleoside analogs such
as fludarabine and gemcitabine are potent radiosensitizers. In animal
experiments, the effect of fludarabine on radiocurability was greater when
fludarabine was combined with fractionated RT than when it was combined with
single-dose RT.77 This implies that the inhibition of sublethal or potentially lethal
Introduction
22
damage repair is a significant mechanism responsible for the enhancement of the
tumor radioresponse to fludarabine.
1.3.3.3. Cell-cycle effects
The third mechanism focuses on a cell-cycle effect. The cytotoxicity of most
chemotherapeutic agents and that of radiation is highly dependent on the phase of
the cell cycle. Both chemotherapeutic agents and radiation are more effective
against proliferating cells than against nonproliferating cells. Among
proliferating cells, cells in the G2 and M phases are the most radiosensitive, and
the cells in the S phase are the most radioresistant.68 Based on this variation in
radiosensitivity over the cell cycle, there exist two strategies for CRT, the use of
chemotherapeutic agents that accumulate cells in a radiosensitive phase or those
that eliminate radioresistant S-phase cells. The latter strategy is related to the
mode of action of nucleoside analogs. Fludarabine and gemcitabine are
incorporated into radioresistant S-phase cells, many of which die by apoptosis.
This preferential removal of S-phase cells therefore contributes to the
radioenhancement effects.
1.3.3.4. Hypoxic cells
Hypoxic cells are 2.5–3.0 times less sensitive to radiation than well-
oxygenated cells.68, 69 Tumors often include hypoxic areas, which is a cause of
radioresistance. Chemotherapeutic agents can improve the RT effect by
eliminating well-oxygenated tumor cells, which leads to tumor reoxygenation,
selectively eliminating hypoxic cells, or sensitizing the hypoxic cells to radiation.
1.3.3.5. Repopulation of tumor cells
The importance of the overall treatment time (OTT) for local tumor control
by RT has been documented in a number of studies of head and neck cancers,
uterine cervical cancer, and esophageal cancer.78-80 Withers and colleagues80
Introduction
23
found that the TCD50 (the radiation dose which yields local control in 50% of
tumors) progressively increased over time if the OTT was prolonged beyond 30
days.80 Their analysis of esophageal and laryngeal squamous cell carcinomas
treated by RT alone showed that the prolongation of OTT significantly reduced
the local control rate.78, 79 One mechanism responsible for this may be the
accelerated repopulation of tumor cells during fractionated RT.
Any approach that reduces or eliminates the accelerated repopulation of
tumor cells improves the efficacy of RT. This is likely to be one of the major
mechanisms by which CT improves local tumor control when given concurrently
with RT. Even a small decrease in repopulation between radiation fractions can
significantly improve the tumor response to fractionated RT. However, most
chemotherapeutic drugs inhibit repopulation not only in the tumor, but also in the
compensatory cell regeneration of normal tissues that occurs during fractionated
RT. Thus, a therapeutic benefit is expected if drugs are tumorselective or if
repopulation is faster in the tumor.
Recently, various molecular targeting drugs have become clinically
available. Several drugs, such as epidermal growth factor receptor (EGFR)
inhibitors, block the membrane receptors of growth factors or interfere with the
signaling pathways involved in cell proliferation. These agents offer another
possible method for inhibiting the accelerated repopulation of tumor cells during
fractionated RT.81, 82
1.3.4. Sequencing of CT and RT
According to the sequencing of CT and RT, CT is designated as either
induction (neoadjuvant) CT, concurrent CT, or adjuvant CT, when it is given
before, during, or after the course of RT, respectively. As clinical trials of
Introduction
24
adjuvant CT following RT have not been systematically studied, the aims and
clinical results of induction CT and concurrent CT are described in the following
paragraphs;
1.3.4.1. Induction chemotherapy
Induction CT has two main objectives,68, 69 one being the eradication of
micrometastases while they contain small numbers of tumor cells, and the other
being to reduce the size of the primary tumor that is to be irradiated. Reducing
the number of clonogenic cells in the tumor increases the probability of tumor
control by RT. In addition, CT induced tumor shrinkage may provide a smaller
target volume for RT, thereby limiting normal tissue damage.
1.3.4.2. Concurrent chemotherapy
For concurrent CRT, CT can act on both systemic and primary lesions.
However, the main objective of concurrent CT is to use CT-RT interactions to
maximize the antitumor effect, even though it inevitably increases the acute
toxicity of the treatment.68-70 Therefore, it should be remembered that the
therapeutic benefit of concurrent CRT only occurs when enhancement of the
tumor response is greater than the toxic effects on critical normal tissues.
For one mode of concurrent CRT, an alternating schedule of CT and RT can
be used.75, 83 For this combination, RT and CT are given alternatively, without a
treatment gap, to minimize excessive toxic effects on normal tissues and to
enhance the tumor response by perturbing cell cycling or reoxygenation. Several
clinical trials involving alternating schedules have yielded promising results.83
Introduction
25
1.4. Synthesis of quinolines
Several synthetic pathways were reported in the literature for the synthesis
of quinoline derivatives;
1.4.1. From aniline derivatives
1.4.1.1. From m-phenylenediamine
7-Amino-4-methyl-quinoline-2-one XLIV was prepared from m-phenylene
diamine XLIII and ethyl acetoacetate.84
1.4.1.2. From substituted anilines
Substituted 2,4-dimethoxyquinolines XLVII were synthesized by cyclo-
condensation of the appropriate substituted anilines XLV with malonic acid and
phosphorus oxychloride to give the 2,4-dichloroquinolines XLVI, followed by
displacement by methoxide ion to give the required quinolines XLVII.85
Introduction
26
1.4.1.3. From acetanilide derivatives
Treatment of acetanilide derivatives XLVIII with Vilsmeier reagent
(DMF/POCl3) gave 2-chloro-3-formylquinolines XLIX in excellent yield 86.
1.4.1.4. From 2-aminobenzonitrile
Warshakoon et al. synthesized the quinoline derivative LI via a Gould-
Jacobs condensation of 2-amino-benzonitrile L with diethyl ethoxymethylene-
malonate. The resulted quinolone LI was then converted to the quinoline
derivative LII.87
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Introduction
27
1.4.1.5. From o-isopropenylaniline
The reaction of o-isopropenylaniline LIII with trifluoro-acetaldehyde ethyl
hemiacetal (TFAE) was carried out in toluene to give 2-trifluoromethylquinoline
derivative LIV and 2-trifluoro-methyl-1,2-dihydroquinoline derivative LV.88
1.4.2. From vanillin
It was reported that alkylation of vanillin LVI with 2-chloroethyl methyl
ether followed by nitration provided compound LVII. Condensation of LVII
with methyl cyanoacetate and subsequent reduction with iron in acetic acid
provided the quinoline derivative LVIII.89
Introduction
28
Also, compound LVII was reacted with malononitrile, followed by
reduction with iron in acetic acid provided the quinoline derivative LIX.89
1.4.3. From 2-aminobenzophenone
Condensation of 2-aminobenzophenone LX with ethyl acetoacetate in the
presence of a catalytic amount of yttrium triflate [Y(SO3CF3)3] at room temp-
erature results in the formation of ethyl 2-methyl-4-phenylquinoline-3-
carboxylate LXI in 92% yield. Similarly, various cyclic ketones such as
cyclopentanone, cyclohexanone and dimedone reacted with 2-aminoaryl ketones
to afford the respective tricyclic quinolines LXII, LXIII and LXIV.90, 91
Introduction
29
1.4.4. Via Diels-Alder reaction
Quinolinediones LXVII were prepared via the Diels-Alder reactions of the
corresponding azadienes LXVI with the desired dienophiles LXV.92
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1.4.5. From cyclohexanone
Ghorab et al. reported that the reaction of aromatic aldehydes with ethyl
cyanoacetate and cyclohexanone in the presence of ammonium acetate yielded
the corresponding tetrahydroquinoline derivatives LXVIII.93
Introduction
30
In addition 2-amino-4-(2-bromophenyl)-5,6,7,8-tetrahydro-quinoline-3-
carbonitrile LXIX was obtained via reaction of 2-bromobenzaldehyde with
malononitrile and cyclohexanone in presence of ammonium acetate.94
1.4.6. From enaminones
The hexahydroquinoline derivative95 LXXI was obtained through reaction of
enaminone LXX with activated cyano olefins in refluxing ethanolic piperidine.96
Also, it was mentioned that the treatment of 5,5-dimethyl-3-(naphthalene-1-
ylamino)-cyclohex-2-enone LXXII with 2-arylidene-malononitriles in the
presence of a catalytic amount of triethylamine resulted in cycloaddition
affording the hexahydroquinolines LXXIII.97
Introduction
31
Additionally, it was reported that the reaction of enaminone LXXII with
ethyl �-cyano-�-ethoxyacrylate by refluxing in ethanol afforded a high yield of 2-
cyano-3-[4,4-dimethyl-2-(naphthalen-1-ylamino)-6-oxo-cyclohex-1-enyl]-acrylic
acid ethyl ester LXXIV which upon heating in glacial acetic acid, afforded the
corresponding quinoline derivative LXXV.97
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Introduction
32
1.4.7. From 3,1-Benzoxazines
Ghorab et al. reported that the reaction of 2-methyl-6-iodo-3,1-benzoxazine
LXXVI with active methylene compounds, namely malononitrile, acetylacetone
or diethylmalonate, gave the corresponding quinoline derivatives LXXVII,
LXXVIII and LXXIX, respectively.98
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1.4.8. From 3-aminocyclohex-2-en-1-one
It has been reported that the 3-aminocyclo-hex-2-en-1-one LXXX99 readily
reacted with 2-benzylidenemalononitrile, in the presence of catalytic amounts of
piperidine, to afford the hexahydroquinoline derivative LXXXI.100
Introduction
33
1.4.9. From Dimedone
The quinoline derivatives LXXXII were prepared via reaction of 2-
arylidenemalononitrile with dimedone and excess of ammonium acetate in acetic
acid as a solvent.101
On the other hand, hexahydroquinoline derivatives LXXXIIIa and
LXXXIIIb were synthesized through condensation of dimedone and aromatic
aldehydes with either methyl 3-aminocrotonate or ethyl acetoacetate in the
presence of ammonium acetate, respectively.102, 103
Introduction
34
1.5. Synthesis of pyrimido[4,5-b]quinolines
Several synthetic pathways were reported in the literature for the synthesis
of pyrimido[4,5-b]quinoline derivatives;
1.5.1. From quinolines
1.5.1.1. From 2-chloro-3-formylquinolines
The 2-oxo-pyrimido[4,5-b]quinoline derivative LXXXV was synthesized by
the condensation reaction of 2-chloro-3-formylquinoline LXXXIV with urea in
the presence of p-toluene sulfonic acid, using microwave irradiation for 5 min.104
1.5.1.2. From 2-aminoquinoline-3-carboxylic acid methyl ester
Cyclization of the quinoline derivative LVIII with formamide at high
temperature gave the corresponding pyrimido[4,5-b]quinoline derivative
LXXXVI.89
Introduction
35
1.5.1.3. From 2-amino-3-cyanoquinolines
Several cyclization reactions were reported for the synthesis of
pyrimido[4,5-b]quinoline derivatives from the 2-amino-3-cyano-quinoline
derivative LXXXVII. When compound LXXXVII was refluxed with either
acetic anhydride, formic acid or formamide, the corresponding pyrimido[4,5-
b]quinoline derivatives LXXXVIII, LXXXIX and XC were obtained,
respectively.97
The 2-thioxo-pyrimido[4,5-b]quinoline XCI was obtained by the reaction of
compound LXXXVII with phenyl isothiocyanate in pyridine.97
Introduction
36
It has been reported that the reaction of compound LXXXVII with
diethyloxalate in ethanol containing sodium ethoxide furnished the 4-ethoxy-
pyrimido[4,5-b] quinoline-2-carboxylic acid ethyl ester XCII.97
The reaction of the quinoline derivatives LXXXVII with benzoylchloride
gave the corresponding 2-phenyl-pyrimido[4,5-b]quinoline derivative XCIII.97
Introduction
37
1.5.2. From pyrimidines
1.5.2.1. From 2,4,6-triaminopyrimidine
Reaction of guanidine nitrate with malononitrile in the presence of sodium
alkoxide in dry ethanol or methanol yielded 2,4,6-triaminopyrimidine XCIV.
Reaction of the pyrimidine derivative XCIV with 2,4-dichlorobenzoic acid, in the
presence of activated copper bronze powder at 180-190°C yielded N-(2,4-
diamino-6-pyrimidino)-4-chloroanthranilic acid XCV. The 2,4-diaminopyrimi-
do[4,5-b]quinoline derivative XCVI was then obtained upon cyclization of XCV
using concentrated sulfuric acid.105
1.5.2.2. From 6-aminopyrimidines
A simple and efficient approach to prepare the pyrimido[4,5-b]quinolines
XCVIII was described in a three components reaction from 6-aminopyrimidines
XCVII, dimedone and aromatic aldehydes.106, 107
AIM OF THE PRESENT INVESTIGATION
Aim of the present investigation
38
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Several structurally novel sulfonamide derivatives have been recently
reported to show substantial anticancer activity in vitro and/or in vivo 22, 108-117. In
order to explain this antitumor activity, several mechanisms were adopted
including carbonic anhydrase inhibition, cell cycle arrest at G1 phase, disruption
of microtubules, and angiogenesis inhibition. The most prominent among these
mechanisms was carbonic anhydrase inhibition.6
On the other hand, quinolines and fused quinoline derivatives are known to
possess several biological activities43-49 including anticancer activity.50-57
Furthermore, it has been reported that some quinoline and pyrimidoquinoline
derivatives containing a sulfonamide moiety exhibit certain anticancer
activity.118, 119
Also, the special properties of the fluorine atom, such as strong
electronegativity, small size and the low polarisability of the C–F bond, can have
considerable impact on the behavior of a molecule in a biological environment.120
The incorporation of fluorine into a drug allows simultaneous modulation of
electronic, lipophilic and steric parameters, all of which can critically influence
both the pharmacodynamic and pharmacokinetic properties of drugs. Bioisosteric
substitution for hydrogen by fluorine is, therefore, an important strategy for
incorporation of a group capable of reinforcing drug–receptor interactions
(electronic modulation), aiding translocation across lipid bilayers or absorption
(lipophilic modulation) and inducing conformational change/blocking
metabolism (steric parameters).121
Aim of the present investigation
39
Based on the above information, and as a continuation of our previously
reported work,122 the present investigation deals with the design, synthesis and in
vitro anticancer evaluation of some new 4-haloarylquinolines and pyrimido[4,5-
b]quinolines, having a free sulfonamide moiety. Recently, some newly
synthesized sulfonamide derivatives were reported to exhibit promising in vitro
cytotoxic activity against human breast cancer cell line (MCF7), in comparison
with doxorubicin and other anticancer drugs.26, 119, 123, 124
As the carbonic anhydrase inhibition is the most prominent mechanism of
the antitumor activity of sulfonamide derivatives, the synthesized compounds
were designed to comply with the previously mentioned pharmacophore of
compounds that may act as CA inhibitors (Figure 3), as this may have a role in
their anticancer activity together with the other anticancer mechanisms of
sulfonamides.
This pharmacophore includes mainly the presence of a sulfonamide moiety
which coordinates with the zinc ion of the active site and the sulfonamide is
attached to an aryl scaffold which is usually a benzene ring. The side chain might
posses a hydrophilic link able to interact with the hydrophilic part of the active
site and a hydrophobic moiety which can interact with the hydrophobic part of
the CA active site.
Figure 6 includes representative examples of the designed compounds,
showing the compliance with the general pharmacophore.
Aim of the present investigation
40
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great compliance to the general pharmacophore of CA inhibitors
Docking of the synthesized compounds will be done on hCA in order to give
an idea if these compounds may act as carbonic anhydrase inhibitors which could
have a role, at least in one part, to their anticancer activity.
The present work reports the design, synthesis, and anticancer evaluation of
the following classes:
Aim of the present investigation
41
i. Hexahydroquinoline derivatives 6, 8, 9, 12, 13 and 16-20, bearing a free
sulfonamide moiety
ii. Pyrimido[4,5-b]quinoline derivatives 7, 10, 11, 14, 15 and 21-25, bearing a
free sulfonamide moiety
Aim of the present investigation
42
On the other hand, the rationale for combining chemotherapy and
radiotherapy is based mainly on two ideas, one being spatial cooperation, which
is effective if chemotherapy is sufficiently active to eradicate subclinical
metastases and if the primary local tumor is effectively treated by radiotherapy.
In this regard, no interaction between radiotherapy and chemotherapy is required.
The other idea is the enhancement of radiation effects by direct enhancement of
the initial radiation damage by incorporating drugs into DNA, inhibiting cellular
repair, accumulating cells in a radiosensitive phase or eliminating radioresistant
phase cells, eliminating hypoxic cells, or inhibiting the accelerated repopulation
of tumor cells. Virtually, all chemotherapeutic agents have the ability to sensitize
cancer cells to the lethal effects of ionizing radiation.67
Consequently, the most two active fluorinated compounds and the most two
active chlorinated compounds, will be selected to be evaluated for their ability to
enhance the cell killing effect of �-irradiation.
THEORETICAL DISCUSSION
Theoretical discussion
43
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Schemes 1-5 illustrate the pathways for the synthesis of the target
compounds.
Scheme 1
Theoretical discussion
44
Scheme 2
Theoretical discussion
45
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Theoretical discussion
46
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Scheme 4
Theoretical discussion
47
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Scheme 5
Theoretical discussion
48
4-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)benzenesulfonamide (3)
Compound 3 was prepared according to the published procedure.122
4-[2-Amino-3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (6a) and 4-[2-amino-4-(4-
chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-
yl]benzenesulfonamide (6b)
Treatment of enaminone 3 with 2-(4-fluorobenzylidene)malononitrile 4a or
2-(4-chlorobenzylidene)malononitrile 4b in presence of a catalytic amount of
triethylamine (TEA), as a basic catalyst, yielded the corresponding
hexahydroquinolines 6a and 6b respectively, via the formation of the
intermediates 5a,b, followed by intramolecular cyclization. The arylidenes 4a
and 4b were prepared by just stirring the corresponding aldehyde with
malononitrile for about 20 min in ethanol containing few drops of TEA at room
temperature.122
Theoretical discussion
49
The N-aryl substituent (benzenesulfonamide) decreases the nucleophilicity
of the enaminone 3 toward 2-arylidene-malononitrile 4a and 4b. The basic
catalyst, TEA; was required to generate the anion of the enaminone 3, thus
facilitating the addition to the unsaturated nitrile 4a and 4b.
The formation of compounds 6a and 6b was proved from their
microanalytical and spectral data. IR spectra of compounds 6a and 6b showed
bands in the range of 3467-3199 cm-1 for (NH2), sharp bands at 2176 & 2174
cm-1 corresponding to the cyano group (C�N) and bands at 1652 & 1645 cm-1 for
(C=O).
1H-NMR spectra of the quinoline derivatives 6a and 6b showed the presence
of singlets at 4.4 & 4.5 ppm for CH, singlets at 5.5 & 5.5 ppm for NH2, and
multiplets for aromatic prortons and SO2NH2 in the range of 7.1-8.0 ppm.
Theoretical discussion
50
Additionally, mass spectra of compound 6a and 6b exhibited molecular ion
peaks at m/z 466 (M+, 39.87%) & m/z 482 (M+, 27.47), with a base peaks at m/z
371, respectively.
4-[5-(4-Fluorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (7a) and 4-[5-(4-
chlorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydropyrimido[4,5-
b]quinolin-10(5H)-yl]benzenesulfonamide (7b)
The pyrimido[4,5-b]quinoline derivatives 7a and 7b were obtained by
refluxing compound 6a or 6b in formic acid. This reaction proceeded via
condensation followed by elimination of two moles of water to give the
pyrimido[4,5-b]quinoline derivatives 7a and 7b, as previously reported in the
literature .125
Theoretical discussion
51
IR spectrum of compound 7a revealed the absence of the band
corresponding to the cyano group and the presence of two bands at 1714 cm-1
and 1647 cm-1, attributed to two C=O of the 4- and 6-oxo groups, respectively.
Additionally, mass spectrum of compound 7a exhibited a molecular ion peak at
m/z 495 (M+1, 0.16%), with a base peak at m/z 101.
Also, IR spectrum of compound 7b revealed the absence of the band
corresponding to the cyano group and the presence of two bands at 1708 cm-1
and 1644 cm-1, corresponding to two C=O of the 4-oxo and 6-oxo groups,
respectively. Additionally, 1H-NMR spectrum of 7b showed the presence of
singlet at 7.9 ppm for NH, singlet at 8.0 ppm for CH=N, and the absence of the
signal corresponding to the 2-amino group.
N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-phenyl)-
1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (8a) and N-[4-(4-
chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-sulfamoylphenyl)-1,4,5,6,7,8-
hexahydroquinolin-2-yl]acetamide (8b)
The starting compounds 6a and 6b were refluxed with acetic anhydride.
Different products were obtained according to the time of the reaction. When
compounds 6a or 6b were refluxed in acetic anhydride for 5 hours, acetylation of
the 2-amino group was carried out affording the corresponding monoacetyl
derivatives 8a and 8b, respectively, instead of the fused pyrimido[4,5-b]quinoline
systems 10a and 10b as previously reported.126
Theoretical discussion
52
The structure of compound 8a was established on the basis of elemental
analysis and spectral data. IR spectrum of compound 8a showed a band at 2212
cm-1 assigned to the cyano group, and two bands at 1725 cm-1 and 1652 cm-1,
attributed to two C=O of the acetyl group and the 5-oxo group, respectively. 1H-
NMR spectrum of compound 8a revealed the acetyl protons as one singlet at 1.5
ppm corresponding to three protons. Furthermore, mass spectrum of compound
8a exhibited a molecular ion peak at m/z 510 (M+2, 1.24%), with a base peak at
m/z 90.
Also, IR spectrum of compound 8b showed a band at 2213 cm-1 assigned to
the cyano group, and two bands at 1720 cm-1 and 1649 cm-1, corresponding to
2C=O of the acetyl group and the 5-oxo group, respectively. In addition, 1H-
NMR spectrum of compound 8b revealed the acetyl protons as one singlet at 1.5
ppm corresponding to three protons.
Theoretical discussion
53
N-Acetyl-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9a) and N-
acetyl-N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9b)
When compounds 6a or 6b was refluxed in acetic anhydride for 10 hours,
acetylation of the 2-amino group was carried out affording the corresponding
diacetyl derivatives 9a and 9b, respectively, instead of the expected fused
pyrimido[4,5-b]quinoline systems 10a and 10b, as previously reported in the
literature.127
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The structure of compound 9a was established on the basis of elemental
analysis and spectral data. IR spectrum of compound 9a showed a band at 2214
Theoretical discussion
54
cm-1 assigned to the cyano group, and two bands at 1727 cm-1 and 1655 cm-1,
corresponding to the carbonyl groups. 1H-NMR spectrum of compound 9a
revealed the acetyl protons as one singlet at 2.4 ppm corresponding to six
protons, and the absence of the signal corresponding to the 2-amino group.
Also, IR spectrum of compound 9b showed a band at 2213 cm-1 assigned to
the cyano group, and two bands at 1728 cm-1 and 1655 cm-1, attributed to the
carbonyl groups. In addition, 1H-NMR spectrum of compound 9b revealed the
acetyl protons as one singlet at 2.4 ppm corresponding to six protons.
Furthermore, mass spectrum of compound 9a exhibited a molecular ion peak at
m/z 566 (M-1, 0.27%), with a base peak at m/z 90.
4-[5-(4-Fluorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (10a) and 4-[5-(4-
chlorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydropyrimido[4,5-
b]quinolin-10(5H)-yl]benzenesulfonamide (10b)
The pyrimido[4,5-b]quinoline derivatives 10a and 10b were obtained by
refluxing compounds 6a or 6b in acetic anhydride for 15 hours, respectively.97
Theoretical discussion
55
The structure of compound 10a was confirmed by the absence of the cyano
group, and the presence of two bands at 1720 cm-1 and 1656 cm-1 for two
carbonyl groups. Additionally, mass spectrum of compound 10a exhibited a
molecular ion peak m/z 508 (M+, 9.66%) and a base peak at m/z 456.
Also, IR spectrum of compound 10b revealed the absence of the band
corresponding to cyano group, and the presence of two bands corresponding to
two carbonyl groups at 1724 cm-1 and 1656 cm-1. Furthermore, 1H-NMR
Theoretical discussion
56
spectrum of compound 10b revealed the 2-methyl group protons as one singlet at
2.4 ppm, corresponding to three protons.
4-[4-Amino-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (11a) and 4-[4-
amino-5-(4-chlorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (11b)
The pyrimido[4,5-b]quinoline derivatives 11a and 11b were obtained by
reaction of compound 6a or 6b with formamide, where cyclization occurred
through elimination of one molecule of water, followed by intramolecular
cyclization.128
The structures of compounds 11a and 11b were established on the basis of
elemental analysis and spectral data. IR spectra of compounds 11a and 11b
revealed the absence of (C�N) bands, which confirms the cyclization and the
formation of the pyrimido[4,5-b]quinoline systems.
Theoretical discussion
57
Also, mass spectra of compounds 11a and 11b showed molecular ion peaks
at m/z 494 (M+1, 0.16%) & m/z 510 (M+, 0.46%) with base peaks at m/z 90 &
87, respectively.
Ethyl N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12a) and Ethyl N-
[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-sulfamoylphenyl)-
1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12b)
The quinoline derivatives 12a and 12b were obtained by refluxing
compound 6a or 6b in triethylorthoformate in the presence of few drops of acetic
anhydride, respectively. The reaction proceeded via the elimination of two moles
of ethanol.119
Theoretical discussion
58
The formation of compounds 12a and 12b was supported by their
microanalytical and spectral data. 1H-NMR spectra showed the presence of a
triplet at 1.2 ppm for the CH3, a quartet at 4.3 ppm of CH2 of the ethyl group and
a singlet corresponding to one proton of the N=CH at 8.7 ppm.
Additionally, mass spectrum of compound 12a showed a molecular ion
peak at m/z 539 (M+, 0.52%) with base peak at m/z 90.
2-Chloro-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (13a) and 2